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DNA Quantitation - Fluorometer Applications

DNA quantitation is an essential step for several life science research protocols.  RNA transcription and transfection, sequencing, cDNA synthesis and cloning are all examples of common DNA techniques which benefit from a defined template concentration.  An inaccurate estimation of the amount of DNA template may cause these techniques to fail.  DNA concentration is often measured by UV absorbance at 260 nm (1A260 = 50 µg/mL) in a 1 cm path length cuvette.  However, greater accuracy can be achieved for quantitating DNA using fluorescent dyes such as Hoechst 33258, a bisbenzimide DNA intercalator that excites in the near UV (350 nm) and emits in the blue region (450 nm).  Hoechst 33258 binds to the AT-rich regions of double stranded DNA and exhibits enhanced fluorescence under high ionic strength conditions.  Fluorescence from Hoechst 33258 is proportional to the concentration of DNA in the sample.